Aryloxyanilide imaging agents

ABSTRACT

The present invention provides a novel radiolabelled aryloxyalinine derivative suitable for in vivo imaging. In comparison to known aryloxyalinine derivative in vivo imaging agents, the in vivo imaging agent of the present invention has better properties for in vivo imaging. The in vivo imaging agent of the present invention demonstrates good selective binding to the peripheral benzodiazepine receptor (PBR), in combination with good brain uptake and in vivo kinetics following administration to a subject.

TECHNICAL FIELD OF THE INVENTION

The present invention concerns in vivo imaging and in particular in vivoimaging of peripheral benzodiazepine receptors (PBR). An aryloxyanilidein vivo imaging agent is provided that binds with nanomolar affinity toPBR, has good uptake into the brain following administration, and whichhas good selective binding to PBR. The present invention also provides aprecursor compound useful in the synthesis of the in vivo imaging agentof the invention, as well as a method for synthesis of said in vivoimaging agent comprising use of said precursor compound, and a kit forcarrying out said method. A cassette for the automated synthesis of thein vivo imaging agent is also provided. In addition, the inventionprovides a radiopharmaceutical composition comprising the in vivoimaging agent of the invention, as well as methods for the use of saidin vivo imaging agent.

DESCRIPTION OF RELATED ART

The peripheral benzodiazepine receptor (PBR) is known to be mainlylocalised in peripheral tissues and glial cells but its physiologicalfunction remains to be clearly elucidated. Subcellularly, PBR is knownto localise on the outer mitochondrial membrane, indicating a potentialrole in the modulation of mitochondrial function and in the immunesystem. It has furthermore been postulated that PBR is involved in cellproliferation, steroidogenesis, calcium flow and cellular respiration.PBR has been associated with a variety of conditions including acute andchronic stress, anxiety, depression, Parkinson's disease, Alzheimer'sdisease, brain damage, cancer (Gavish et al Pharm Rev 1999; 51: 629),Huntington's disease (Meβmer and Reynolds Neurosci Lett 1998; 241:53-6), asthma (Pelaia et al Gen Pharmacol 1997; 28(4): 495-8),rheumatoid arthritis (Bribes et al Eur J Pharmacol 2002; 452(1):111-22), atherosclerosis (Davies et al J Nucl Med 2004; 45: 1898-1907)and multiple sclerosis (Banati et al Brain 2000; 123: 2321). PBR mayalso be associated with neuropathic pain, Tsuda et al having observedactivated microglia in subjects with neuropathic pain (TINS 2005; 28(2):101-7).

Positron emission tomography (PET) imaging using the PBR selectiveligand, (R)-[¹¹C]PK11195 provides a generic indicator of central nervoussystem (CNS) inflammation. Despite the successful use of(R)-[¹¹C]PK11195, it has its limitations. It is known to have highprotein binding, and low specific to non-specific binding. The role ofits radiolabelled metabolites is not known and quantification of bindingrequires complex modelling. There have been efforts to provide compoundshaving high affinity and selectivity for PBR to enable improvedmeasurement of PBR in the CNS.

Aryloxyalinine derivatives have been proposed that have high affinityfor PBR, as well as high selectivity for PBR over the centralbenzodiazepine receptor (CBR) (Chaki et al Eur J Pharmacol 1999; 371:197-204). [¹¹C]-DAA1106 and [¹⁸F]-FE-DAA1106 are PET radioligands basedon these aryloxyalinine compounds. These PET radioligands are taught inU.S. Pat. No. 6,870,069, and have been studied in humans (Ikomo et al JCereb Blood Flow Metab 2007; 27: 173-84 and Fujimura et al J Nuc Med2006; 47: 43-50). Alternative radiofluorinated DAA1106 derivatives aretaught in WO 2007/074383. Alternative ¹¹C-labelled DAA1106 derivativesare described in WO 2007/036785. Radioiodinated DAA1106 is described inEP 1854781, and by Zhang et al (J Med Chem 2007; 50: 848-55). Thechemical structures of [¹¹C]-DAA1106, [¹⁸F]-FE-DAA1106 and[¹²³I]-DAA1106 are as follows:

However, the kinetic properties of these compounds are not ideal for invivo imaging such that their application to quantitative studies arebelieved to be limited.

More recently, a compound known as PBR06 has been reported as havingimproved properties for in vivo imaging as compared with theabove-described compounds. The structure of this compound is as follows:

Although metabolised quite rapidly in the periphery, the metabolites ofPBR06 do not penetrate the blood-brain barrier (Briard et al J Med Chem2009; 52: 688-699). In contrast to the earlier compounds, almost theentire signal coming from the brain is from intact PBR06. This enablesthe concentration of PBR in the brain to be accurately determined. PBR06is therefore regarded as a promising in vivo imaging agent.

However, and as presented in the present specification, the ratio ofuptake of PBR06 in tissues that have a relatively high expression of PBR(i.e. olfactory bulb) as compared with background tissues (i.e.striatum) is less than optimal for in vivo imaging. There is also scopeto improve the proportion of intact compound in the brain.

There is therefore a need for alternative in vivo imaging agents havingimproved properties for in vivo imaging as compared with PBR06, i.e.higher specific uptake in PBR-expressing tissues, and/or higherproportion of intact compound in the brain.

SUMMARY OF THE INVENTION

The present invention provides a novel radiolabelled aryloxyalininederivative suitable for in vivo imaging. In comparison to knownaryloxyalinine derivative in vivo imaging agents, the in vivo imagingagent of the present invention has better properties for in vivoimaging. The in vivo imaging agent of the present invention demonstratesimproved specific binding to the peripheral benzodiazepine receptor(PBR), in addition to having good brain uptake and favourable in vivokinetics.

DETAILED DESCRIPTION OF THE INVENTION Imaging Agent

In one aspect, the present invention provides an in vivo imaging agentof Formula I:

-   -   wherein:    -   A¹ is —CR¹R²—(CH₂)_(n)— wherein R¹ and R² are independently        selected from hydrogen, fluoro, or C₁₋₃ alkyl, and n is 0, 1 or        2;    -   A² is —CH₂—, —O— or —O—CH₂—; or,    -   -A¹-A²- is —CH═CH—.

An “in vivo imaging agent” in the context of the present invention is aradiolabelled compound suitable for in vivo imaging. The term “in vivoimaging” as used herein refers to those techniques that non-invasivelyproduce images of all or part of the internal aspect of a subject.Examples of such in vivo imaging methods are single photon emissioncomputed tomography (SPECT) and positron emission tomography (PET).

Unless otherwise specified, the term “alkyl” alone or in combination,means a straight-chain or branched-chain alkyl radical containingpreferably from 1 to 3 carbon atoms. Examples of such radicals include,methyl, ethyl, and propyl.

Examples of some in vivo imaging agents of the invention are as follows:

A¹ of Formula I is preferably —CR¹R²—(CH₂)_(n)—, most preferably—(CH₂)_(m)— wherein m is 1, 2 or 3, and especially preferably 1 or 2. A²of Formula I is preferably —CH₂— or —O—. Especially preferably -A¹-A²-is selected from —CH₂—CH₂—, —CH₂—O— and —CH₂—CH₂—O—. Preferred in vivoimaging agents of the invention are in vivo imaging agents 1-4, mostpreferably in vivo imaging agents 1-3, especially preferably in vivoimaging agents 1 and 2 and most especially preferably in vivo imagingagent 1.

Example 11 describes the rat biodistribution model that was used tocompare in vivo imaging agents of the invention with PBR06. Evaluationof the OB:striatum uptake as compared with PBR06 revealed that in vivoimaging agent 1 was taken up relatively more in the OB as compared withthe striatum (see FIG. 1 herein), and in vivo imaging agents 2 and 3were comparable with PBR06 (see FIGS. 2 and 3). As OB is known toexpress higher levels of PBR compared with other areas of rat brain (see“Handbook of Substance Abuse” by Tarter, Ammerman and Ott; Springer1998:398-99) the ratio OB:striatum is a measure of specificity of testcompound uptake.

Example 12 describes the assay used to evaluate the amount of intacttest in vivo imaging agent in rat brain at 60 minutes post-injection. Invivo imaging agents 1-4 demonstrated the same favourable metabolismprofile as PBR06, i.e. a high proportion of radioactivity in the brainat 60 minutes post-injection was found to be intact test compound. Invivo imaging agents 1 and 2 were found to have an even higher proportionintact in the brain at 60 minutes post-injection as compared with PBR06.

In vivo imaging agents of the present invention are shown herein to havesuperior properties for in vivo imaging of PBR as compared with knownsuch agents.

Method for Preparation

In a further aspect, the present invention provides a method for thepreparation of the in vivo imaging agent as defined herein, wherein saidmethod comprises reacting a suitable source of ¹⁸F with a precursorcompound of Formula II:

-   -   wherein A¹ and A² are as suitably and preferably defined herein        for the in vivo imaging agent of Formula I, and LG is a leaving        group.

A “precursor compound” comprises a non-radioactive derivative of the invivo imaging agent, designed so that chemical reaction with a convenientchemical form of ¹⁸F occurs site-specifically; can be conducted in theminimum number of steps (ideally a single step); and without the needfor significant purification (ideally no further purification), to givethe desired in vivo imaging agent. Such precursor compounds aresynthetic and can conveniently be obtained in good chemical purity.

The term “a suitable source of ¹⁸F” means ¹⁸F in a chemical form that isreactive with a substituent of the precursor compound such that ¹⁸Fbecomes covalently attached thereby forming the desired in vivo imagingagent.

Broadly speaking, the step of “reacting” the precursor compound withsaid suitable source of ¹⁸F involves bringing the two reactants togetherunder reaction conditions suitable for formation of the desired in vivoimaging agent in as high a radiochemical yield (RCY) as possible. Somemore detailed routes are presented in the experimental section below.

The term “leaving group” refers to an atom or group of atoms that leavesa molecule with a pair of electrons in heterolytic bond cleavage,usually to be replaced by a nucleophile. A leaving group can be an anionor a neutral molecule. Preferred leaving groups (LG) are mentionedbelow.

Okubu et al (2004 Bioorg. Med. Chem.; 12: 423-38) describe methods toobtain non-radioactive aryloxyanilide compounds. Synthetic schemes toobtain aryloxyanilide compounds are also described by Briard et al (J.Med. Chem. 2008; 51; 17-31), Wilson et al (Nuc. Med. Biol. 2008; 35;305-14), and Zhang et al (J. Med. Chem. 2007; 50: 848-55). These priorart methods can be easily adapted to obtain a precursor compound ofFormula II.

Scheme I below is a generic reaction scheme to obtain non-radioactivestandards, and precursor compounds suitable for preparation of the invivo imaging agents of the present invention:

In the above reaction scheme, LG is a leaving group as defined herein,and R* represents the fused bicyclic ring structure that includes the Aring, as comprised in both Formula I and Formula II above. Reduction ofthe nitro group in commercially-available 2-nitrobiphenyl ether (a) byhydrogenation gives the corresponding aniline (b). Reductive alkylationwith an aromatic aldehyde (c) gives the benzylamine (d). Reaction withthe appropriate acetyl (e) results in the desired non-radioactivestandard or precursor compound.

Labelling with ¹⁸F is achieved by nucleophilic displacement of theleaving group LG from the precursor compound of Formula II. Preferredleaving groups (LG) include chloride, bromide, iodide, tosylate,mesylate, and triflate, with bromide and tosylate being most preferred.The precursor compound of Formula II may be labelled in a one stepreaction wherein the suitable source of ¹⁸F is [¹⁸F]-fluoride.[¹⁸F]fluoride (¹⁸F

) for radiofluorination reactions is normally obtained as an aqueoussolution from the nuclear reaction ¹⁸O(p,n)¹⁸F and is made reactive bythe addition of a cationic counterion and the subsequent removal ofwater. Suitable cationic counterions should possess sufficientsolubility within the anhydrous reaction solvent to maintain thesolubility of ¹⁸F

. Therefore, counterions that have been used include large but softmetal ions such as rubidium or caesium, potassium complexed with acryptand such as Kryptofix™, or tetraalkylammonium salts. A preferredcounterion is potassium complexed with a cryptand such as Kryptofix™because of its good solubility in anhydrous solvents and enhanced ¹⁸F

reactivity.

To ensure that radiofluorination takes place at a particular site, theprecursor compound may need to be selectively chemically protected.Protecting groups have been discussed above.

The precursor compound is ideally provided in sterile, apyrogenic form.It can accordingly be used for the preparation of a pharmaceuticalcomposition comprising the in vivo imaging agent together with abiocompatible carrier suitable for mammalian administration. Theprecursor compound is also suitable for inclusion as a component in akit for the preparation of such a pharmaceutical composition.

In a preferred embodiment, the precursor compound is provided insolution and as part of a kit, or of a cassette designed for use in anautomated synthesis apparatus. These aspects are discussed in moredetail below in relation to additional aspects of the invention.

In another preferred embodiment, the precursor compound is bound to asolid phase. The precursor compound is preferably supplied covalentlyattached to a solid support matrix. In this way, the desired productforms in solution, whereas starting materials and impurities remainbound to the solid phase. As an example of such a system, precursorcompounds for solid phase electrophilic fluorination with ¹⁸F-fluorideare described in WO 03/002489, and precursor compounds for solid phasenucleophilic fluorination with ¹⁸F-fluoride are described in WO03/002157.

Preferably, the method of the present invention is automated for ease ofperformance.

Precursor Compound

The precursor compound as suitably and preferably described above inrelation to the method of the invention itself forms an additionalaspect of the present invention.

Radiopharmaceutical Composition

In a yet further aspect, the present invention provides a“radiopharmaceutical composition”, which is a composition comprising thein vivo imaging agent of the invention, together with a biocompatiblecarrier in a form suitable for mammalian administration.

The “biocompatible carrier” is a fluid, especially a liquid, in whichthe in vivo imaging agent is suspended or dissolved, such that theradiopharmaceutical composition is physiologically tolerable, i.e. canbe administered to the mammalian body without toxicity or unduediscomfort. The biocompatible carrier is suitably an injectable carrierliquid such as sterile, pyrogen-free water for injection; an aqueoussolution such as saline (which may advantageously be balanced so thatthe final product for injection is either isotonic or not hypotonic); anaqueous solution of one or more tonicity-adjusting substances (e.g.salts of plasma cations with biocompatible counterions), sugars (e.g.glucose or sucrose), sugar alcohols (e.g. sorbitol or mannitol), glycols(e.g. glycerol), or other non-ionic polyol materials (e.g.polyethyleneglycols, propylene glycols and the like). The biocompatiblecarrier may also comprise biocompatible organic solvents such asethanol. Such organic solvents are useful to solubilise more lipophiliccompounds or formulations. Preferably the biocompatible carrier ispyrogen-free water for injection, isotonic saline or an aqueous ethanolsolution. The pH of the biocompatible carrier for intravenous injectionis suitably in the range 4.0 to 10.5.

Suitable and preferred embodiments of the in vivo imaging agent whencomprised in the radiopharmaceutical composition of the invention are asdefined above.

The radiopharmaceutical composition may be administered parenterally,i.e. by injection, and is most preferably an aqueous solution. Such acomposition may optionally contain further ingredients such as buffers;pharmaceutically acceptable solubilisers (e.g. cyclodextrins orsurfactants such as Pluronic, Tween or phospholipids); pharmaceuticallyacceptable stabilisers or antioxidants (such as ascorbic acid, gentisicacid or para-aminobenzoic acid). Where the in vivo imaging agent of theinvention is provided as a radiopharmaceutical composition, the methodfor preparation of said in vivo imaging agent may further comprise thesteps required to obtain a radiopharmaceutical composition, e.g. removalof organic solvent, addition of a biocompatible buffer and any optionalfurther ingredients. For parenteral administration, steps to ensure thatthe radiopharmaceutical composition is sterile and apyrogenic also needto be taken.

Kit and Cassette

In a preferred embodiment, the method for the preparation of the in vivoimaging agent of the invention is carried out by means of a kit, orusing a cassette that can plug into an automated synthesiser. These kitsand cassettes in turn form further aspects of the invention, and areparticularly convenient for the preparation of the radiopharmaceuticalcomposition of the invention as defined herein.

The kit of the invention comprises the precursor compound of theinvention in a sealed container. The “sealed container” preferablypermits maintenance of sterile integrity and/or radioactive safety, plusoptionally an inert headspace gas (e.g. nitrogen or argon), whilstpermitting addition and withdrawal of solutions by syringe. A preferredsealed container is a septum-sealed vial, wherein the gas-tight closureis crimped on with an overseal (typically of aluminium). Such sealedcontainers have the additional advantage that the closure can withstandvacuum if desired e.g. to change the headspace gas or degas solutions.

Suitable and preferred embodiments of the precursor compound whenemployed in the kit of the invention are as already described herein.

The precursor compound for use in the kit may be employed under asepticmanufacture conditions to give the desired sterile, non-pyrogenicmaterial. The precursor compound may alternatively be employed undernon-sterile conditions, followed by terminal sterilisation using e.g.gamma-irradiation, autoclaving, dry heat or chemical treatment (e.g.with ethylene oxide). Preferably, the precursor compound is provided insterile, non-pyrogenic form. Most preferably the sterile, non-pyrogenicprecursor compound is provided in the sealed container as describedabove.

Preferably, all components of the kit are disposable to minimise thepossibilities of contamination between runs and to ensure sterility andquality assurance.

In another aspect, the present invention provides a cassette which canbe plugged into a suitably adapted automated synthesiser for thesynthesis of the in vivo imaging agent of the invention.[¹⁸F]-radiotracers radiotracers in particular are now often convenientlyprepared on an automated radiosynthesis apparatus. There are severalcommercially-available examples of such apparatus, including Tracerlab™and Fastlab™ (both available from GE Healthcare). The radiochemistry isperformed on the automated synthesis apparatus by fitting the cassetteto the apparatus. The cassette normally includes fluid pathways, areaction vessel, and ports for receiving reagent vials as well as anysolid-phase extraction cartridges used in post-radiosynthetic clean upsteps.

The cassette for the automated synthesis of the in vivo imaging agent ofthe invention comprises:

-   (i) a vessel containing a precursor compound as defined herein; and-   (ii) means for eluting the vessel with a suitable source of ¹⁸F, as    defined herein.

The cassette may additionally comprise:

-   (iii) an ion-exchange cartridge for removal of excess ¹⁸F; and    optionally,-   (iv) a cartridge for deprotection of the resultant radiolabelled    product to form an in vivo imaging agent as defined herein.

The reagents, solvents and other consumables required for the automatedsynthesis may also be included together with a data medium, such as acompact disc carrying software, which allows the automated synthesiserto be operated in a way to meet the end user's requirements forconcentration, volumes, time of delivery etc.

Methods of Use

In a yet further aspect, the present invention provides an in vivoimaging method for determining the distribution and/or the extent of PBRexpression in a subject comprising:

-   -   (i) administering to said subject an in vivo imaging agent as        defined herein;    -   (ii) allowing said in vivo imaging agent to bind to PBR in said        subject;    -   (iii) detecting by an in vivo imaging procedure signals emitted        by ¹⁸F of said in vivo imaging agent;    -   (iv) generating an image representative of the location and/or        amount of said signals; and,    -   (v) determining the distribution and extent of PBR expression in        said subject wherein said expression is directly correlated with        said signals emitted by said ¹⁸F.

For the in vivo imaging method of the invention, suitable and preferredaspects of the in vivo imaging agent are as defined earlier in thespecification.

“Administering” the in vivo imaging agent is preferably carried outparenterally, and most preferably intravenously. The intravenous routerepresents the most efficient way to deliver the in vivo imaging agentthroughout the body of the subject, and therefore also across theblood-brain barrier (BBB) and into contact with PBR expressed in saidsubject. The in vivo imaging agent of the invention is preferablyadministered as the pharmaceutical composition of the invention, asdefined herein. In an alternative embodiment, the administration stepcan be understood as a preliminary step carried out before the in vivoimaging method itself, such that step (i) can be defined as providing asubject to whom the in vivo imaging agent has been pre-administered.

Following the administering step and preceding the detecting step, thein vivo imaging agent is allowed to bind to PBR. For example, when thesubject is an intact mammal, the in vivo imaging agent will dynamicallymove through the mammal's body, coming into contact with various tissuestherein. Once the in vivo imaging agent comes into contact with PBR, aspecific interaction takes place such that clearance of the in vivoimaging agent from tissue with PBR takes longer than from tissuewithout, or with less PBR. A certain point in time will be reached whendetection of in vivo imaging agent specifically bound to PBR is enabledas a result of the ratio between in vivo imaging agent bound to tissuewith PBR versus that bound in tissue without, or with less PBR. Ideally,this ratio is 2:1 or greater.

The “detecting” step of the method of the invention involves detectionof signals emitted by the ¹⁸F by means of a positron-emission tomography(PET) detector. This detection step can also be understood as theacquisition of signal data.

The “generating” step of the method of the invention is carried out by acomputer which applies a reconstruction algorithm to the acquired signaldata to yield a dataset. This dataset is then manipulated to generateimages showing the location and/or amount of signals emitted by said¹⁸F. The signals emitted directly correlate with the expression of PBRsuch that the “determining” step can be made by evaluating the generatedimage.

The “subject” of the invention can be any human or animal subject.Preferably the subject of the invention is a mammal. Most preferably,said subject is an intact mammalian body in vivo. In an especiallypreferred embodiment, the subject of the invention is a human. The invivo imaging method may be used to study PBR in healthy subjects, or insubjects known or suspected to have a pathological condition associatedwith abnormal expression of PBR (a “PBR condition”). The in vivo imagingagents of the invention are particularly suited to in vivo imaging PBRexpression in the central nervous system (CNS).

In an alternative embodiment, the in vivo imaging method of theinvention may be carried out repeatedly during the course of a treatmentregimen for said subject, said regimen comprising administration of adrug to combat a PBR condition. For example, the in vivo imaging methodof the invention can be carried out before, during and after treatmentwith a drug to combat a PBR condition. In this way, the effect of saidtreatment can be monitored over time. PET imaging is particularlysuitable for this embodiment. PET has excellent sensitivity andresolution, so that even relatively small changes in a lesion can beobserved over time, which is advantageous for treatment monitoring. PETscanners routinely measure radioactivity concentrations in the picomolarrange. Micro-PET scanners now approach a spatial resolution of about 1mm, and clinical scanners about 4-5 mm.

Preferably, said method relates to the in vivo imaging of a subjectknown or suspected to have a PBR condition, and therefore is useful aspart of a method for the diagnosis of said condition. The in vivoimaging method of the invention may therefore comprise the further step(vi) of attributing the distribution and extent of PBR expression todiagnose whether said subject is suffering from a PBR condition.Examples of such PBR conditions where in vivo imaging would be of useinclude neuropathologies such as Parkinson's disease, multiplesclerosis, Alzheimer's disease and Huntington's disease whereneuroinflammation is present. Other PBR conditions that may be usefullyimaged with the compounds of the invention include neuropathic pain,arthritis, asthma, atherosclerosis, a range of malignant diseasesincluding but not limited to colorectal cancer and breast cancer, andalso a range of mood disorders including but not limited to bipolardisorder, schizophrenia, anxiety and post-traumatic stress disorder.

In another aspect, the present invention provides the in vivo imagingagent as defined herein for use in the in vivo imaging method assuitably and preferably defined herein.

In a yet further aspect, the present invention provides the in vivoimaging agent as defined herein for the manufacture of aradiopharmaceutical composition as defined herein for use in the in vivoimaging method as suitably and preferably defined herein.

The invention is now illustrated by a series of non-limiting examples.

BRIEF DESCRIPTION OF THE EXAMPLES

Example 1 describes the synthesis of the direct labelling precursor forin vivo imaging agent 1.

Example 2 describes the synthesis of the direct labelling precursor forin vivo imaging agent 2.

Example 3 describes the synthesis of the direct labelling precursor forin vivo imaging agent 3.

Example 4 describes the synthesis of the direct labelling precursor forin vivo imaging agent 4.

Example 5 describes the radiofluorination method used to obtain in vivoimaging agents 1-4.

Example 6 describes the synthesis of a non-radioactive standard for invivo imaging agent 1.

Example 7 describes the synthesis of a non-radioactive standard for invivo imaging agent 2.

Example 8 describes the synthesis of a non-radioactive standard for invivo imaging agent 3.

Example 9 describes the synthesis of a non-radioactive standard for invivo imaging agent 4.

Example 10 describes the in vitro assay used to evaluate the affinity ofnon-radioactive standards of the imaging agents of the invention forPBR.

Example 11 describes the animal model used to determine biodistributionof the imaging agents of the invention following intravenousadministration.

Example 12 describes the assay used to evaluate the metabolism of theimaging agents of the invention following intravenous administration.

LIST OF ABBREVIATIONS USED IN THE EXAMPLES

-   ° C. degrees celsius-   aq aqueous-   BGO Bismuth germanate-   DCM dichloromethane-   DMF dimethyl formamide-   DMSO dimethyl sulfoxide-   EtOAc ethyl acetate-   g grams-   h hours-   K_(i) concentration of a compound required for half maximum    inhibition-   MBq megabequerels-   mg milligrams-   min minutes-   ml millilitres-   mM millimolar-   mmol millimoles-   n number of experiments-   NMR nuclear magnetic resonance-   PBR peripheral benzodiazepine receptor-   rpm revolutions per minute-   TEA triethylamine-   TLC thin layer chromatography-   Tris tris(hydroxymethyl)aminomethane-   UV ultraviolet

EXAMPLES Example 1 Synthesis of the Direct Labelling Precursor for InVivo Imaging Agent 1 Example 1(i) 2-Aminodiphenyl ether

2-Nitrodiphenyl ether (16 g, 74 mmol) in methanol (250 ml) was shakenwith palladium on charcoal (1.6 g) under an atmosphere of hydrogen at20-50° C. for 30 min. There was a rapid uptake of hydrogen and adetectable exotherm 20-50° C. with the temperature rapidly rising beforecooling. Shaking was stopped for short periods to control thetemperature from rising above 50° C. The reaction was then filteredthrough celite and concentrated in high vacuum to give 2-aminodiphenylether as an oil (13.5 g, 72.9 mmole, 98%) that crystallized on standingto give a buff solid.

¹ H NMR (CDCl₃) 300 MHz δ 3.82 (2H, brm, NH₂), 6.7-7.1 (7H, m, ArH) 7.33(2H, m, ArH).

¹³C NMR (CDCl₃) 75 MHz δ•116.41, 117.03, 118.70 (2C), 120.22, 122.57,124.85, 129.65 (2C), 138.70, 142.97, 157.43.

Example 1(ii)N-(2,3-Dihydro-benzofuran-7-ylmethyl)-N-(2-phenoxyphenyl)-amine

2-Aminodiphenyl ether (1 g, 5.4 mmol) was treated with2,3-Dihydro-benzofuran-7-carbaldehyde (1 g, 7.02 mmol) and toluene (10ml) and heated at reflux for 4 h under an atmosphere of nitrogen withvigorous stirring. The solution became yellow and homogeneous. Thereaction was then concentrated in vacuum to remove the toluene, cooledto 0° C., diluted with methanol (15 ml), and treated with sodiumborohydride (612 mg 16 mmol) in portions over a period of 20 min. Thereaction was then allowed to warm to room temperature and stirred for afurther 30 min. 2N hydrochloric acid (5 ml) was added and the reactionstirred for a further 30 min. The reaction was then concentrated invacuum to a gum and 10% aq potassium carbonate (50 ml) added. Theproduct was then recovered by extraction into ethyl acetate (50 ml), theextract was dried over magnesium sulphate and concentrated in vacuum toa gum. The gum was chromatographed on a 120 g silica column in agradient of 10-30% ethyl acetate in petrol. The main fast runningfraction,N-(2,3-Dihydro-benzofuran-7-ylmethyl)-N-(2-phenoxyphenyl)-amine wascollected as a gum (1.2824 g, 4.04 mmole, 74.9%) that crystallized onstanding.

¹H NMR CDCl₃ 300 MHz, δ•3.19(2H, t, CH₂Ph), 4.35 (2H, s, CH₂N) 4.51 (2H,t, CH₂O) 4.87 (1H, brs, NH), 6.6, −7.31(12H, m ArH).

¹³C NMR CDCl₃, 75 MHz δ•29.69, 42.63, 71.11, 112.01, 116.82, 117.27,119.34, 120.31, 122.48, 123.64, 126.96, 129.55, 140.50, 143.07, 157.71,157.83.

Example 1(iii)2-Bromo-N-(2,3-dihydrofuran-7-ylmethyl)-N-(2-phenoxyphenyl)-acetamide

N-(2,3-Dihydrobenzofuran-7-ylmethyl)-N-(2-phenoxyphenyl)amine (0.5 g,1.57 mmol) in dichloromethane (10 ml) was cooled to 0° C. and treatedwith bromoacetyl chloride (272 mg, 1.73 mmol) and triethylamine (175 mg,1.73 mmol) and stirred for 1 h under an atmosphere of nitrogen. Thereaction was diluted with dichloromethane (50 ml) and washed with 5Nhydrochloric acid (20 ml) to remove the triethylamine and aqueouspotassium carbonate (20 ml) to remove excess bromoacetyl chloride. Theorganic layer was separated, dried over magnesium sulphate andconcentrated in high vacuum to give2-bromo-N-(2,3-dihydrofuran-7-ylmethyl)-N-(2-phenoxyphenyl)acetamide.(661 mg, 1.51 mmole, 96%).

¹H NMR (CDCl₃) 300 MHz, δ•3.06(2H, m, CH₂Ph), 3.8 (2H, d, d CH₂O),4.2-4.4 (2H, d, q, CH₂Br) 4.7, (1H, d, CHN), 5.12, (1H, d, CHN),6.62-7.32 (12H, m, ArH).

¹³C NMR (CDCl₃) 75 MHz δ 27.81, 29.68, 46.78, 70.89, 118.04, 119.36,120.26, 123.06, 124.13, 124.17, 129.82, 153.48, 155.59, 158.55, 166.64.

Example 2 Synthesis of the Direct Labelling Precursor for In VivoImaging Agent 2 Example 2(i)N-(benzo[1,3]dioxol-4-ylmethyl)-N-(2-phenyloxy-phenyl)amine

A mixture of 2-phenoxy-phenylamine (410 mg, 2.22 mmol) and2,3-(methylenedioxy)-benzaldehyde (500 mg, 3.33 mmol) was heated at 90°C. for 2 h under nitrogen. The reaction was cooled to 0° C. and MeOH (4mL) was added, followed by NaBH₄ (253 mg, 6.70 mmol) in portions over 20min. The mixture was stirred at room temperature for 24 h. Formic acid(0.4 mL was added and the mixture stirred for 15 min. The solvents wereremoved in vacuo, the residue quenched with saturated aqueous NaHCO₃ (50mL), extracted with DCM (2×30 mL), dried over MgSO₄, filtered andsolvents removed in vacuo. The crude material was purified by silica gelchromatography eluting with petroleum spirit (A) and ethyl acetate (B)(5% B, 80 g, 4.0 CV, 60 mL/min) to afford 360 mg (51%) ofN-(benzo[1,3]dioxol-4-ylmethyl)-N-(2-phenyloxy-phenyl) amine as a whitesolid.

¹ H NMR (300 MHz, CDCl₃) δ 4.35 (2H, d, J=5.2 Hz, ArCH ₂), 4.66 (1H, s,NH), 5.88 (2H, s, OCH ₂O), 6.60-7.10 (10H, m, ArH), 7.24-7.34 (2H, m,ArH).

Example 2(ii)N-(benzo[1,3]dioxol-4-ylmethyl)-2-Bromo-N-(2-phenyloxy-phenyl)acetamide

To a solution ofN-(benzo[1,3]dioxol-4-ylmethyl)-N-(2-phenyloxy-phenyl)amine (0.18 g,0.58 mmol) dissolved in DCM (4 mL) was added triethylamine (0.24 g, 2.32mmol, 0.32 mL). The reaction was cooled to 0° C. and bromoacetylchloride (0.18 g, 1.16 mmol, 0.10 mL) was added. The mixture was stirredat room temperature for 2 h. LC-MS indicated starting material andproduct (1:1). Further triethylamine (0.24 g, 2.32 mmol, 0.32 mL) andbromoacetyl chloride (0.18 g, 1.16 mmol, 0.10 mL) were added and stirredat room temperature for 2 h. The solvents were removed in vacuo, theresidue quenched with water (10 mL), extracted with DCM (2×20 mL), driedover MgSO₄, filtered and solvents removed in vacuo. The crude materialwas purified by silica gel chromatography eluting with DCM (A) and MeOH(B) (1% B, 80 g, 2 CV, 60 mL/min). The impure product was furtherpurified by silica gel chromatography eluting with DCM (A) and EtOAc (B)(1-5% B, 80 g, 4.5 CV, 60 mL/min) to afford 120 mg (47%) ofN-(benzo[1,3]dioxol-4-ylmethyl)-2-Bromo-N-(2-phenyloxy-phenyl)acetamideas a colourless oil.

¹ H NMR (300 MHz, CDCl₃) δ 3.76 (1H, d, J=11 Hz, BrCH), 3.82 (1H, d,J=11 Hz, BrCH), 4.72 (1H, d, J=14 Hz, ArCH), 5.13 (1H, d, J=14 Hz,ArCH), 5.63 (1H, d, J=1 Hz, OCHO), 5.79 (1H, d, J=1 Hz, OCHO), 6.65-7.40(12H, m, ArH).

LC-MS: m/z calcd for C₂₂H₁₈BrNO₄ 440.3; found, 441.9 (M+H)+.

Example 3 Synthesis of the Direct Labelling Precursor for In VivoImaging Agent 3 Example 3(i)N-(2,3-Dihydrobenzo[1,4]dioxinyl-5-ylmethyl)-N-(2-phenoxyphenyl)-amine

2-Aminodiphenyl ether (1 g, 5.4 mmol) was treated with2,3-Dihydro-benzo[1,4]dioxinyl-5-aldehyde. (885 mg, 5.4 mmol) andtoluene (10 ml) and heated at reflux for 4 h under an atmosphere ofnitrogen with vigorous stirring. The solution became yellow andhomogeneous. The reaction was then concentrated in vacuum to remove thetoluene, cooled to 0° C., and diluted with methanol (25 ml) and treatedwith sodium borohydride (1 g, pellet) with continuous stirring. Thereaction was then allowed to warm to room temperature overnight when awhite crystalline solid had precipitated. The solid collected byfiltration was(N-(2,3-Dihydrobenzo-[1,4]dioxinyl-5-ylmethyl)-N-(2-phenoxyphenyl)-amine,1.128 g, 3.56 mmole, 66%.

¹ H NMR CDCl₃ 300 MHz, δ 4.16, (4H, s, CH₂—Ox2), 4.35 (2H, d, CH₂N),4.68 (1H, t, NH), 6.6-7.1 (10H, m ArH), 7.29, (2H, t, ArH).

¹³C NMR CDCl₃, 75 MHz, δ•42.6, 64.06, 64.21, 112.18, 116.25, 117.09,119.57, 120.62, 120.79, 124.97, 128.00, 129.58, 140.55, 141.00, 142.97,143.33, 157.78.

Example 3(ii)N-(2,3-Dihydro-benzo-[1,4]dioxinyl-5-ylmethyl)-2-bromo-N-(2-phenoxyphenyl)acetamide

N-(2,3-Dihydro-benzo[1,4]dioxin-5-ylmethyl)-N-(2-phenoxyphenyl)-amine(pure) (0.5 g, 1.5 mmol) in dichloromethane (20 ml) was treated withbromoacetyl chloride (259 mg, 1.65 mmol) and triethylamine (168 mg, 1.65mmol) at 0° C. for 1 h. The reaction was then diluted withdichloromethane (50 ml) and washed with 2N hydrochloric acid (20 ml) toremove the triethylamine and 10% aq potassium carbonate solution toremove excess fluoroacetyl chloride. The organic layer was separateddried over magnesium sulphate and concentrated in high vacuum to2-bromo-N-(2,3-dihydro-benzo-[1,4]dioxinyl-5-ylmethyl)-N-(2-phenoxyphenyl)acetamide,637 mg, 1.40 mmole, 93%.

¹H NMR CDCl₃. 300 MHz, δ 3.77(2H, dd, CH₂Br), 3.85-4.2 (4H, m, CH₂Ox2),4.75, and 5.15(1H, d, together CH₂N), 6.65-7.4 (12H, m, ArH).

¹³C NMR CDCl₃ 75 MHz, δ 27.77, 46.08, 63.95, 64.02, 116.68, 119.39,120.66, 122.97, 123.00, 124.78, 124.28, 124.70, 129.50, 129.90, 130.40,131.00 141.98, 143.16, 153.58, 155.50, 166.53.

Example 4 Synthesis of the Direct Labelling Precursor for In VivoImaging Agent 4 Example 4(i)N-(2,2,-Dimethyl-2,3-dihydrobenzofuran-7-ylmethyl)-N-(2-phenoxyphenyl)amine

A mixture of 2-phenoxy-phenylamine (350 mg, 1.89 mmol) and2,2-dimethyl-2,3-dihydro-1-benzofuran-7-carbaldehyde (500 mg, 2.83 mmol)was heated at 90° C. for 2 h under nitrogen. The reaction was cooled to0° C. and MeOH (4 mL) was added, followed by NaBH₄ (216 mg, 5.70 mmol)in portions over 20 min. The mixture was stirred at room temperature for24 h. Formic acid (0.4 mL was added and the mixture stirred for 15 min.The solvents were removed in vacuo, the residue quenched with saturatedaqueous NaHCO₃ (50 mL), extracted with DCM (2×30 mL), dried over MgSO₄,filtered and solvents removed in vacuo. The crude material was purifiedby silica gel chromatography eluting with petroleum spirit (A) and ethylacetate (B) (5-10% B, 80 g, 3 CV, 60 mL/min) to afford 560 mg (86%) ofN-(2,2,-Dimethyl-2,3-dihydrobenzofuran-7-ylmethyl)-N-(2-phenoxyphenyl)amine as a colourless oil.

¹H NMR (300 MHz, CDCl₃) δ 1.43 (6H, s, C(CH ₃)₂), 2.98 (2H, s, ArCH ₂),4.32 (2H, s, NCH ₂), 4.79 (1H, s, NH), 6.60-7.10 (10H, m, ArH), 7.30(2H, m, ArH).

Example 4(ii)2-Bromo-N-(2,2,-Dimethyl-2,3-dihydrobenzofuran-7-ylmethyl)-N-(2-phenoxyphenyl)acetamide

To a solution ofN-(2,2,-Dimethyl-2,3-dihydrobenzofuran-7-ylmethyl)-N-(2-phenoxyphenyl)amine (0.20 g, 0.58 mmol) dissolved in DCM (2 mL) was addedtriethylamine (0.24 g, 2.32 mmol, 0.32 mL). The reaction was cooled to0° C. and bromoacetyl chloride (0.18 g, 1.16 mmol, 0.10 mL) was added.The mixture was stirred at room temperature for 2 h. LC-MS indicatedstarting material and product (1:1). Further triethylamine (0.24 g, 2.32mmol, 0.32 mL) and bromoacetyl chloride (0.18 g, 1.16 mmol, 0.10 mL)were added and stirred at room temperature for 2 h. The solvents wereremoved in vacuo, the residue quenched with water (10 mL), extractedwith DCM (2×20 mL), dried over MgSO₄, filtered and solvents removed invacuo. The crude material was purified by silica gel chromatographyeluting with petroleum spirit (A) and EtOAc (B) (30% B, 80 g, 2.5 CV, 60mL/min). The impure product was further purified by silica gelchromatography eluting with DCM (A) and EtOAc (B) (1-5% B, 80 g, 5 CV,60 mL/min) to afford 170 mg (63%) of2-Bromo-N-(2,2,-Dimethyl-2,3-dihydrobenzofuran-7-ylmethyl)-N-(2-phenoxyphenyl)acetamide as a colourless oil.

¹H NMR (300 MHz, CDCl₃) δ 1.14 (3H, s, CH ₃), 1.31 (3H, s, CH ₃), 2.83(1H, d, J=15 Hz, ArCH), 2.90 (1H, d, J=15 Hz, ArCH), 3.74 (1H, d, J=11Hz, BrCH), 3.80 (1H, d, J=11 Hz, BrCH), 4.59 (1H, d, J=14 Hz, NCH), 5.22(1H, d, J=14 Hz, NCH), 6.65-7.40 (12H, m, ArH)

LC-MS: m/z calcd for C₂₅H₂₄BrNO₃ 466.4; found, 467.9 (M+H)+

Example 5 Radio Fluorination of the Precursor Compounds of Examples 1-4to Obtain In Vivo Imaging Agents 1-4 5(i) Drying [¹⁸F] Fluoride

To ¹⁸F-fluoride supplied in 15 μl water, a further 200 μl water wasadded, and the fluoride drawn into a COC vessel. In the presence ofKryptofix (2 mg, 5.3×10-6 moles), dissolved in 0.5 ml acetonitrile and0.1 M potassium hydrogen carbonate solution (50 μl, 5×10-6 moles), the¹⁸F-fluoride was dried at 110° C./30 minutes under a flow of nitrogen(0.3 L/min for ˜20 minutes followed by 0.1 L/min when the long tap wasopened. The flow was turned up to 0.5 L/min when the risk of splashingwas no longer (for ˜10 min) and then cooled to room temperature.

5(ii) Radio Fluorination

To the dry residue obtained in step 5(i) was added 0.7 mg of theselected precursor compound in 1 ml acetonitrile and the reaction heatedin a sealed system at 100° C./10 minutes. After cooling, the reactionmixture was transferred to an N46 vial and the COC vial rinsed with 1.5ml water. The washings were transferred to same glass vial. The prep(whose total volume was ˜2.5 mL) was drawn onto the prep HPLC and theHPLC cut diluted in ˜15 ml water prior to loading onto a Sep-Pak tC18light (pre-conditioned with 2.5 ml ethanol and 5 ml water). The Sep-Pakwas then eluted with 0.5 ml ethanol (collected into a P6 vial) followedby 4.6 ml Dulbecco's phosphate buffered saline collected into the sameP6 vial. The RCP was measured by HPLC.

Example 6 Synthesis of a Non-Radioactive Standard for In Vivo ImagingAgent 1 Example 6(i)N-(2,3-Dihydrobenxofuran-7-ylmethyl)-2-fluoro-N-(2-phenoxyphenyl)acetamide

N-(2,3-Dihydro-benzofuran-7-ylmethyl)-N-(2-phenoxyphenyl)-amine (0.5 g,1.78 mmol) in DCM (20 ml) was treated with fluoroacetyl chloride (199mg, 1.96 mmol) and TEA (199 mg, 1.96 mmol) at 0° C. for 1 h. Thereaction was then diluted with DCM (50 ml) and washed with 2Nhydrochloric acid (20 ml) to remove the TEA and 10% aq potassiumcarbonate to remove fluoroacetyl chloride. The organic layer separateddried over magnesium sulphate and concentrated in high vacuum to giveN-(2,3-Dihydrobenxofuran-7-ylmethyl)-2-fluoro-N-(2-phenoxyphenyl)acetamide455 mg, 1.61 mmole, 91%.

¹ H NMR in CDCl₃, 300 mHz, δ 2.9-3.13 (2H, m, CH₂Ph), 4.22, and 4.38(1H,q, together CH₂O) 4.7 and 4.9 (1H, q, together CH₂F), 4.8 and 5.1 (1H,d, together CH₂N), 6.63-7.5 (12H, m, ArH).

-   ¹³C NMR in CDCl₃, 75 MHz, δ 26.73, 43, 31, 68.03, 77.00, 114.00,    115.00 116.39, 117.32, 120.23, 121.34, 124.00, 126.36, 126.94,    127.45, 150.84, 152.58, 155.76, 164.11, 164.38.

Example 7 Synthesis of a Non-Radioactive Standard for In Vivo ImagingAgent 2 Example 7(i)N-(benzo[1,3]dioxol-4-ylmethyl)-2-Fluoro-N-(2-phenyloxy-phenyl)acetamide

To a solution ofN-(benzo[1,3]dioxol-4-ylmethyl)-N-(2-phenyloxy-phenyl)amine (0.16 g,0.50 mmol) dissolved in DCM (2 mL) was added TEA (0.20 g, 2.00 mmol,0.28 mL). The reaction was cooled to 0° C. and fluoroacetyl chloride(0.10 g, 1.00 mmol, 0.07 mL) was added. The mixture was stirred at roomtemperature for 1 h. The solvents were removed in vacuo, the residuequenched with water (10 mL), extracted with DCM (2×20 mL), dried overMgSO₄, filtered and solvents removed in vacuo. The crude material waspurified by silica gel chromatography eluting with DCM (A) and MeOH (B)(1-5% B, 80 g, 4.0 CV, 60 mL/min) to afford 160 mg (84%) ofN-(benzo[1,3]dioxol-4-ylmethyl)-2-Fluoro-N-(2-phenyloxy-phenyl)acetamideas a yellow oil. The structure was confirmed by ¹H NMR (300 MHz, CDCl₃)δ 4.67 (1H, d, J=2 Hz, FCH), 4.83 (1H, d, J=2 Hz, FCH), 4.74 (1H, d,J=14 Hz, ArCH), 5.12 (1H, d, J=14 Hz, ArCH), 5.63 (1H, d, J=1 Hz, OCHO),5.79 (1H, d, J=1 Hz, OCHO), 6.66-6.88 (6H, m, ArH), 6.96-7.06 (2H, m,ArH), 7.12-7.38 (4H, m, ArH).

¹⁹F NMR (282 MHz, CDCl₃) δ −226.8, −227.0, −227.2.

LC-MS: m/z calcd for C₂₂H₁₈FNO₄ 379.4; found, 380.1 (M+H)+.

Example 8 Synthesis of a Non-Radioactive Standard for In Vivo ImagingAgent 3 Example 8(i) 2-fluoro-5N-(2,3-Dihydro-benzo-[1,4]dioxinyl-5-ylmethyl)-N-(2-phenoxyphenyl)acetamide

N-(2,3-Dihydro-benzo[1,4]-5-ylmethyl)-N-(2-phenoxyphenyl)-amine (0.5 g,1.5 mmol) in DCM (20 ml) was treated with fluoroacetyl chloride (168 mg,1.65 mmol) and TEA (168 mg, 1.65 mmol) at 0° C. for 1 h. The reactionwas then diluted with DCM (50 ml) and washed with 2N hydrochloric acid(20 ml) and 10% aq potassium carbonate solution. The organic layer wasseparated dried over magnesium sulphate and concentrated in high vacuumto a gum. The gum was then chromatographed on 120 g silica column in agradient of 15-40% ethyl acetate in petrol. The major fractions wascollected to give2-fluoro-N-(2,3-Dihydro-benzo-[1,4]dioxinyl-5-ylmethyl)-N-(2-phenoxyphenyl)acetamide(0.538 g, 1.43 mmole, 95%.)

¹ H NMR in CDCl₃, 300 MHz, δ 3.8-4.1 (4H, M, CH₂Ox2), 4.65, and 4.81(1H, d, d, together, CH₂F), 4.8, and 5.1 (1H, d, d, together CH₂N),6.6-7.4, (12H m, ArH).

¹³C NMR in CDCl₃, 75 MHz, δ 45.2, 63.72, 63.83, 77.41, 79.75, 116.58,119.16, 120.41, 122.98, 123.00, 124.16, 124.30, 129.00, 129.74, 130.00,141.91, 143.05, 153.71, 155.23, 166.49, 166.75.

Example 9 Synthesis of a Non-Radioactive Standard for In Vivo ImagingAgent 4 Example 9(i)—N-(2,2,-Dimethyl-2,3-dihydrobenzofuran-7-ylmethyl)-2-Fluoro-N-(2-phenoxyphenyl)acetamide

To a solution ofN-(2,2,-Dimethyl-2,3-dihydrobenzofuran-7-ylmethyl)-N-(2-phenoxyphenyl)amine (0.20 g, 0.58 mmol) dissolved in DCM (2 mL) was added TEA(0.24 g, 2.32 mmol, 0.32 mL). The reaction was cooled to 0° C. andfluoroacetyl chloride (0.11 g, 1.16 mmol, 0.08 mL) was added. Themixture was stirred at room temperature for 1 h. The solvents wereremoved in vacuo, the residue quenched with water (10 mL), extractedwith DCM (2×20 mL), dried over MgSO₄, filtered and solvents removed invacuo. The crude material was purified by silica gel chromatographyeluting with DCM (A) and MeOH (B) (1% B, 80 g, 2 CV, 60 mL/min) toafford 170 mg (72%) ofN-(2,2,-Dimethyl-2,3-dihydrobenzofuran-7-ylmethyl)-2-Fluoro-N-(2-phenoxyphenyl)acetamide as a pale yellow oil.

¹H NMR (300 MHz, CDCl₃) δ 1.12 (3H, s, CH ₃), 1.30 (3H, s, CH ₃), 2.83(1H, d, J=15 Hz, ArCH), 2.90 (1H, d, J=15 Hz, ArCH), 4.58 (1H, d, J=14Hz, NCH), 4.65 (1H, s, FCH), 4.81 (1H, s, FCH), 5.22 (1H, d, J=14 Hz,NCH), 6.65-7.40 (12H, m, ArH).

¹⁹F NMR (282 MHz, CDCl₃) δ −226.6, −226.8, −227.0

LC-MS: m/z calcd for C₂₅H₂₄FNO₃ 405.5; found, 406.1 (M+H)+.

Example 10 In Vitro Potency Assay

Affinity for PBR was screened using a method adapted from Le Fur et al(Life Sci. 1983; USA 33: 449-57). The compounds tested were PBR06, andin vivo imaging agents 1-4.

Each test compound (dissolved in 50 mM Tris-HCl, pH 7.4, 10 mM MgCl₂containing 1% DMSO) competed for binding to Wistar rat heart PBR against0.3 nM [³H] PK-11195. The reaction was carried out in 50 mM Tris-HCl, pH7.4 10 mM MgCl₂ for 15 minutes at 25° C.

Each test compound was screened at 6 different concentrations over a300-fold range of concentrations around the estimated K_(i). The K_(i)values for PBR06 and in vivo imaging agents 1-4 were found to be 0.28nM, 0.31 nM 2.03 nM, 1.14 nM and 2.66 nM, respectively.

Example 11 In Vivo Biodistribution Method

PBR06 (synthesised according to Briard et al J Med Chem 2009; 52:688-699) and in vivo imaging agents 1-3 were tested in an in vivobiodistribution model and their respective biodistributions compared.

Adult male Wistar rats (200-300 g) were injected with 1-3 MBq of testcompound via the lateral tail vein. At 2, 10, 30 or 60 min (n=3) afterinjection, rats were euthanised and tissues or fluids were sampled forradioactive measurement on a gamma counter.

FIGS. 1-3 illustrate the ratio of uptake of PBR06 and in vivo imagingagents 1-3, respectively, in OB compared to uptake in the striatum.

Example 12 Metabolism Assay

Brain tissue samples were collected from adult male Wistar rats (200-300g) at 60 minutes after injection of test in vivo imaging agent. Thesesamples were then processed via solvent extraction (see below) toextract the ¹⁸F-labelled parent along with any ¹⁸F-labelled metabolites,before introduction to the HPLC.

Brain (minus cerebellum+medulla pons) was homogenized with 10 mls ofice-cold Acetonitrile (5000 rpm for 5 mins) to extract all the¹⁸F-labelled species. The resulting supernatant was then evaporated todryness (rotary evaporation at 40° C.), concentrated in 2.5 mls ofmobile phase, filtered and 1 ml was injected onto the HPLC.

The HPLC set up for ¹⁸F analysis was connected to a dual BGO radio & UVdetector. A μBondapak C18 prep column was used having dimensions 7.8×300mm; 10 μm; 125 Å. An isocratic elution system was used using between30-40% water and between 60-70% acetonitrile. The flow rate was 3ml/min. The aqueous to organic phase ratio was varied for each test invivo imaging agent to obtain a parent peak at retention time at oraround 10 min±2 min.

At 60 minutes post-injection the percentage of radioactivity in thebrain representing intact test compound was 90%, 93% 92%, and 82%,respectively, for PBR06, and in vivo imaging agents 1-3.

What is claimed is: 1)-19) (canceled) 20) A method for the preparationof an in vivo imaging agent of Formula I:

wherein: A¹ is —CR¹R²—(CH₂)_(n)— wherein R¹ and R² are independentlyselected from hydrogen, fluoro, or C₁₋₃ alkyl, and n is 0, 1 or 2; A² is—CH₂—, —O— or —O—CH₂—; or, -A¹-A²- is —CH═CH— said method comprisesreacting a suitable source of ¹⁸F with a precursor compound of FormulaII:

wherein A¹ and A² are as defined for the in vivo imaging agent ofFormula I, and LG is a leaving group. 21) The method as defined in claim20 wherein LG is selected from chloride, bromide, iodide, tosylate,mesylate, and triflate. 22) The method as defined in claim 20 whereinsaid suitable source of ¹⁸F is [¹⁸F]fluoride. 23) The method as definedin claim 20 wherein said method is automated. 24) A precursor compoundof Formula II as defined in the method of claim
 20. 25) A kit forcarrying out the method as defined in claim 20 comprising a precursorcompound of Formula II in a sealed container. 26) A cassette forcarrying out the method as defined in claim 23, said cassettecomprising: (i) a vessel containing the precursor compound of FormulaII; and, (ii) means for eluting the vessel with a suitable source of the¹⁸F. 27) The cassette as defined in claim 26 which additionallycomprises: (iii) an ion-exchange cartridge for removal of excess ¹⁸F.